Heparinase activity

Heparinase Activity. Several assays were used to follow heparinase activity. These assays followed (1) the disappearance of heparin, (2) the appearance of heparin degradation products, or (3) the loss of the physiological function of heparin in anticoagulation. The basis of these assays and explanations as to when they are routinely used are listed below. 

Studies of the effect of pH on HA heparinase activity and stability determined that the activity maximum occurs at pH 5.8, while the stability maximum occurs at pH 7.0. 

The CNBr-activated Sepharose 4B support (1 g dry weight) was swelled in 25 mL of hydrochloric acid (0.001 Af), and then washed with 100 mL of 0.5M NaCl, 0.1 M NaHC03 buffer at pH 8.3. To this support 5.5 mL of hydroxylapatite-purified heparinase (0.2 mg/mL protein with an activity of 88 units/mg protein in 0.2M phosphate buffer at pH 7.0) and 60 mg of heparin were added. The mixture was shaken overnight at 4°C, after which the beads were washed and blocked overnight at 4°C with a solution of lysine at pH 8.2 in 0.5M NaCl, 0.1M NaHC03 buffer solution. This support showed an uptake of 87% of the protein and an immobilization of 91% of the heparinase activity. 

One of the critical factors in our research has been the adaptation and use of multiple assays to follow heparinase activity. Particularly important were assays (e.g., Azure A) used in monitoring the fermentation and early stages of purification. By utilizing three different approaches for assaying heparin (disappearance of heparin, appearance of reaction products, and disappearance of heparin s biological activity), the occurrence of any arti-... 

Using the preceding technique, the authors identified that they should focus on improving the fermentation step, and the affinity chromatography recovery steps. For example, they noted that by increasing the yield from the affinity chromatography step to 90% led to a decrease in the cost by 25%. Also, an increase to 2000 mg/mL of expression level of active, refolded heparinase I leads to a 12% reduction in cost. These numbers may be compared to the base case. 

R.J. Linhardt, A. Grant, C.L. Cooney, and R. Langer, "Differential Anti-Coagulant Activity of Heparin Fragments Prepared Using Microbial Heparinase", J. Biol. Chem., 257, 7310, 1982 ... 

The first step in characterizing the heparinase binding rate to the catalyst particles is to establish experimental conditions where neither enzyme denaturation or external mass transfer are important. This can be accomplished by controlling the duration of immobilization, the mixing rate, and the catalyst particle size. In the absence of diffusional limitations and enzyme denaturation effects, the disappearance of enzymatic activity from the bulk phase equals the rate at which the enzyme binds to the catalyst particle. The molar conservation equation for heparinase in the bulk phase is given by... 

Normally, the immobilization of heparinase to agarose catalyst particles is terminated after 4-5 h because greater than 85% of the initial heparinase is bound (49). Based on a cyanate ester stability study, the cyanate ester concentration drops to only 88% of its initial value. For modeling purposes, the cyanate ester concentration was assumed constant. In addition, because of its small size relative to the large molecular weight cutoff (1.5 x 106 daltons) of the catalyst particle, cyanogen bromide (MW 106) should diffuse rapidly into the particle and uniformly activate the matrix. 

Figure 2. Results of a typical fermentation on complex medium showing heparin (Q), heparinase specific activity (Q), and ary cell weight (A) as a function of time as determined in a 2-L fermentor (15).

An affinity column was prepared by immobilizing partially hydrolyzed poly(vinyl sulfate) on epoxy-activated Sepharose (25). Heparinase (HA purified) was bound to this column, and was released at either high or low pH (11 or 4) with 5-10% total activity recovery and up to 500% enrichment (21). 

IEF also was applied towards the HA-purified enzyme to obtain highly pure heparinase. The enzyme was loaded onto a prefocused acrylamide gel at pH 7.0. After IEF, the enzymatic activity was recovered at pH 8.5 0.5. The resulting enzyme had a specific activity of about 5000 units/mg protein, having undergone an enrichment of 50-fold (21). 

The purification of heparinase has been followed by SDS gel electrophoresis. The crude sonicate gave more than 20 major bands the HA purified enzyme, 3 major bands and the IEF purified enzyme, 2 major bands. A summary of the specific activities, protein recoveries, and enzyme purity obtained using our purification procedures is listed in Table I. 

Detailed studies have been performed on the activity and stability of heparinase. Hydroxylapatite-purified heparinase (HA) is stable to freezethawing and freeze-drying, with 90 and 87% recovered activity, respectively. Highly purified heparinase requires the addition of BSA or polylysine (0.05%) and glycerol (7.5%) to permit 100% activity recovery on freezethawing. 

Heparinase Immobilization. Heparinase has been immobilized on a variety of supports, with a widely differing degree of success. The best results have been obtained on Sepharose and polyacrylamide. Low levels of activity recovery occurred on PHEMA. The other supports tested gave either no activity recovery or only barely detectable levels of activity (Table II). 

To check several of the immobilization methods, chondroitinase ABC (from Proteus vulgaris) was used as a control. A summary of the activity recoveries of immobilized heparinase and chondroitinase is listed in Table II. 

Figure 4. Activity profile of heparinase. Key A, specific activity of native enzyme, and Q> specific activity of the Sepharose-immobilized enzyme.

In Vitro Studies on Immobilized Heparinase. Initial experiments have been conducted to test the effectiveness of immobilized heparinase in removing heparin in vitro. Controls consisted of Sepharose-heparinase that was denatured by heating at 100°C for 30 min. In one set of experiments, both active and denatured immobilized heparinase were loaded into two columns, both with a 1.5-mL bed volume. Solutions of heparin, BSA (60... 

We have just begun a series of experiments in which citrated rabbit blood, heparinized at a level of 10 units/mL (153 units/mg), was passed through a Sepharose-heparinase column (0.5mL) at a flow rate of 0.5 mL/min (Figure 8). After 5 min the blood leaving the bottom of the column was sampled and assayed for heparin by whole blood clotting time and Factor Xa heparin assays. In the active column, 50% of the heparin was removed. However, when the same heparinized blood was treated with a control column, less than a 5% decrease in anticoagulant activity was observed. 

While our studies on heparinase production and purification have been encouraging, less success has been achieved in the immobilization procedures (Table II). Studies are in progress to understand better the important parameters in immobilization procedures and in establishing new supports. Initial results indicate that a noncharged support with a high surface area is best (Table II). Additionally, our preliminary evidence is that high levels of heparinase (> 1 mg/mL) and the presence of substrate in the immobilization reaction enhance the recovery of immobilized enzyme activity. 

Figure 8. In vitro heparin removal from citrated rabbit blood by passing heparin through a 500-1xL Sepharose-heparinase column at a flow rate of 0.5 mL/min. The anticoagulant activity as measured bu both Factor Xa and whole blood clotting time is shown for the untreated blood and for the blood cycled through either the natured or denatured Sepharose-heparinase...

Heparinase immobilized on a negatively charged support probably will result in substrate repulsion, and thus reduced activity due to the strong negative charge of heparin. This result may, in fact, explain the apparent poor activities of some of the immobilized heparinase preparations listed in Table II. 

The initial tests of immobilized heparinase on heparinized blood were limited to short time periods (< 5 min). At later times, apparent decreases in heparin levels were observed in the control columns, although at a slower rate than with the active column. This effect may be due to blood damage occurring on the column. Such damage by Sepharose is not unexpected (37), and research is in progress to use either a different support with better blood compatibility or a Sepharose column with a lower bed-to-blood volume ratio. 

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